Our overarching goal is to investigate the importance of protein post-translational modification (PTM) networks in cancer. The signaling system defined by the PTM lysine methylation has recently emerged as a potential key regulator of cancer pathways. However, in comparison to enzymes like protein kinases that have well-established roles in tumorigenesis, the mechanisms by which lysine methyltransferase (KMT) enzymes contribute to cancer are poorly understood. We believe that characterizing novel KMTs involved in the post-translational control of cellular proteins in cancer-relevant signaling networks can provide novel insights into the basic mechanisms of cellular transformation and identify new therapeutic targets for cancer treatment. SMYD3 is a KMT whose expression is up-regulated in a wide range of human cancers. We and others have shown that transient overexpression of SMYD3 can promote the tumorigenic potential of several cancer cell lines. However, knowledge of the endogenous substrates of SMYD3 and the overall mode of action of this enzyme in cells and in vivo is still obscure. We hypothesize that SMYD3 promotes the proliferation and the survival of cancer cells by methylating and regulating key cellular effectors including non-histone proteins. We will first test this idea by exploring the mode of action of SMYD3 in cancer in vivo, which has not previously been done. In Aim 1, we will use a novel conditional mutant Smyd3 allele to test the hypothesis that loss of SMYD3 inhibits tumorigenesis. Because SMYD3 levels are more specifically elevated in tumors with oncogenic RAS, we will focus on mouse models of lung adenocarcinoma and pancreas ductal carcinoma, two fatal human cancers in which the RAS pathway is often activated. In Aim 2 we will analyze the molecular mechanisms of SMYD3 action in cancer cells. We recently identified the MEKK2 (MAP3K2) kinase as a SMYD3 substrate. MEKK2 belongs to the same family as RAF proteins and mediates cellular responses to various growth factors, including EGF, to regulate signaling networks such as the JNK and NF-kB pathways. We will test the hypothesis that SMYD3-mediated methylation of MEKK2 constitutes a new mechanism by which cancer cells respond to stress and growth factors. The goal of Aim 3 is to identify new substrates of SMYD3 using a novel chemical biological- proteomic strategy we have developed for proteome-wide discovery of functionally-relevant SMYD3 substrates. The role of the most promising targets in regulation of cancer cell pathways will be investigated using a combination of molecular approaches and mouse genetics. Based on accumulating evidence implicating a role for SMYD3 in cancer, a number of pharmaceutical companies and academic laboratories are developing SMYD3 inhibitors. Our studies have the capacity to identify SMYD3 as an attractive therapeutic target in many cancer types affecting a wide range of patients. Furthermore, the identification of key SMYD3 molecular targets may identify novel biomarkers and promising new candidate therapeutic targets in cancer.